Immunomodulation with novel pharmaceutical compositions

ABSTRACT

The synthesis and use of a novel class of tumor necrosis factor (TNFα) inhibitors and immunomodulators are provided. Examples are those having the structures:  
                 
 
wherein a, b and c are integers from 0 to 12, X equals NH or CHNH 2 , R 1  and R 2  each is a hydrogen or a C 1  to C 20  aliphatic; aliphatic amine; an alicyclic; aromatic; heterocycle; and halogenated forms thereof; and  
                 
         wherein, a, b and c are integers from 0 to 12; R 1 , R 2 , R 3 , and R 4  each is a hydrogen or a C 1  to C 20 ; aliphatic amine; an alicyclic; aromatic; a heterocycle; and halogenated forms thereof.

FIELD OF THE INVENTION

The present invention relates to the synthesis and use of a novel classof tumor necrosis factor (TNFα) inhibitors and immunomodulators. Thesecompounds have pharmacological applications as well as uses in assaysrelating to TNFα and other involved cytokines. As pharmaceuticals, thesecompounds are used to treat inflammatory, infectious, autoimmune orother proliferative diseases and conditions related to the unwantedpresence or activity of TNFα and/or one or more other involvedcytokines, alone or in combination with other agents.

BACKGROUND OF THE INVENTION

Exuberant production of pro-inflammatory cytokines has been implicatedin the pathogenesis of numerous inflammatory and autoimmune diseases.Secretion of TNFα is a primary event in the initiation of theinflammatory cascade (Brennan F. M., et. al. Lancet, 1989, 2:244-7;Haworth C, et. al. Eur. J. Immunol. 1991, 21:2575-2579) and directlycontributes to the initiation and maintenance of these diseases. Otherpro-inflammatory cytokines also play a role, including interleukin 1β(IL-1β), IL-6, IL-8, IL-12 nitric oxide (NO), IFN-γ and granulocytemacrophage-colony stimulating factor (GM-CSF), while anti-inflammatorycytokines such as IL-10 may reduce disease. Cells of the immune system,macrophages in particular, secrete many of these cytokines in responseto activating stimuli. Blocking pro-inflammatory cytokines and TNFα inparticular has been shown to improve the symptoms and progression of avariety of inflammatory diseases. Thus, inhibition of either theproduction or the activity of TNFα and/or modulation of other cytokinesactive in the disease is an appealing therapeutic target for thetreatment of inflammatory, infectious, autoimmune and otherproliferative diseases and conditions.

Many inflammatory diseases have been treated with steroids,methotrexate, immunosuppressive drugs including cyclophosphamide,cyclosporine, azathioprine and leflunomide, nonsteroidalanti-inflammatory agents including aspirin, acetaminophen and cox-2inhibitors, gold agents and anti-malarials. These drugs have a varietyof disadvantages such as adverse reactions and lack of efficacy. Newanti-TNFα biologic therapies have emerged that give a faster onset ofrelief and improved efficacy. However, these protein-based therapiesalso suffer drawbacks including adverse side effects such as injectionsite reactions, rash, upper respiratory infections, autoimmune disordersand serious infections. Another shortcoming of these biologic therapiesis their required route of administration, intravenous (IV) orsubcutaneous (SC), as opposed to more convenient and compliant oral ordermal routes. Accordingly, a need still exists for the development ofnovel small molecule compositions that inhibit TNFα and/or modulate theexpression of one or more other cytokines that can be used to treatinflammatory, infectious, autoimmune and other proliferative diseasesand conditions.

The pathogenesis of rheumatoid arthritis (RA), a chronic progressiveinflammatory autoimmune disorder, is mediated by cytokines. Severalcytokines, including TNFα (Di Giovine F. S., et. al. Ann. Rheum. Dis.1988, 47:768-772) IL-1 (Rooney M., et. al. Rheumatol Int. 1990,10:217-219), IL-10 (Arend W. P. et. al. Arthritis. Rheum. 1990,30:305-315), and GM-CSF (Firestein G. S., et. al. J. Exp. Med. 1988,168:1573-1586; Xu W. D., et. al. J. Clin. Invest. 1989, 83:876-882), areupregulated in the joints of RA patients with active disease. Elevatedlevels of TNFα and IL-1β can contribute to joint swelling seen inexperimental animal models of arthritis and human RA (Thorbecke, G. J.et. al. Proc. Natl. Acad. Sci., 1992, 89:7375-7379; Chu C. Q., et. al.Arth. Rheum. 1991, 34:1125-1132; Farahat M. N., et. al. Ann. Rheum. Dis.1993, 52:870-875; Brennan F. M., et. al. Eur J Immunol. 1992,22:1907-1912; Elliott M. J., et. al. Baillieres Clin. Rheumatol. 1995,9:633-652). Anti-TNFα therapy using both antibodies and fusion proteinsagainst TNFα has been shown to reduce the symptoms of collagen-inducedarthritis in mice (Williams, R. O., et. al. Proc. Natl. Acad. Sci.,1992, 89:9784-9788; Wooley P. H., et. al. J Immunol, 1993,151:6602-6607; Mori L., et. al. J Immunol, 1996, 157:3178-3182) and inhuman clinical settings (Elliott M. J., et. al. Arth Rheum. 1993,36:1681-1690; Elliott M. J., et. al. Lancet, 1994, 344:1105-1110;Elliott M. J., et. al. Lancet, 1994; 344:1125-1127; Moreland L. W., et.al. N. Engl J. Med. 1997, 337:141-147; Moreland L. W., et. al. J.Rheumatol. 1996, 23:1849-55; Rankin E. C., et. al. Br. J. Rheumatol.1995, 34:334-42; Sander O., et. al. Arth. Rheum. 1996, 39 (suppl.):S242(Abstract)). Recently, anti-IL-1β therapy using a recombinant IL-1receptor antagonist has been shown efficacious in combination withmethotrexate in treating RA patients (Cohen S., et. al. Arth. Rheum.2002, 46:614-624). In experimental animal models of arthritis,inhibition of NO production (Brahn E., et. al. J. Rheumatol. 1998,25:1785-1793), blockage of GM-CSF activity (Cook A. D., et. al. Arth.Res. 2001, 3:293-298) or treatment with recombinant IL-10 (ananti-inflammatory cytokine) (Tanaka T., et. al. Inflamm. Res. 1996;45:283-288) reduced arthritic symptoms. These studies underscore thecontribution of individual cytokines to the pathogenesis of RA and implythat therapies that target more than one cytokine or a selective subsetof cytokines may be more efficacious.

Therapies that target TNFα have been shown to be effective in otherinflammatory diseases in humans and in animal models of human disease.Anti-TNFα therapy has been used to effectively treat Crohn's disease, achronic inflammatory bowel disease. Infliximab (Remicade, Centocor,Inc.), an intravenously administered monoclonal antibody to TNFα, hasbeen approved for commercial use in refractory Crohn's disease. Mikedresults have been seen in patients receiving Etanercept (Enbrel, AmgenCorp.), an injectable TNFα receptor fusion protein. In a small study,patients responded favorably and had decreased inflammatory markers(D'Haens G. et. al. Am J Gastroenterol. 2001, 96:2564-2568) followingtreatment with this drug. In a different study, Etanercept was found tobe ineffective for the treatment of patients with moderate to severeCrohn's disease (Sandbom W. J., et. al. Gastroenterology 2001,121:1088-1094). The reasons for lack of efficacy could be mechanistic,dose-related or may indicate that the biologic drug did not reach thetarget cell population. Another TNFα modulator, the small moleculethalidomide, which both down-regulates TNFα production and inhibitsangiogenesis, has been shown to relieve experimentaliodoacetamide-induced colitis in rats (Kenet G., et. al. Isr. Med.Assoc. J. 2001, 9:644-648). Additionally, early findings in a long-termtreatment protocol involving children and young adults treated withthalidomide suggest that treatment resulted in decreased Crohn's diseaseactivity (Facchini S., et. al. J Pediatr. Gastroenterol. Nutr. 2000,32:178-181).

Spondyloarthropathies (SpA) are a group of related disorders withvarying clinical symptoms including spondylitis, synovitis, psoriaticarthritis and subclinical gut inflammation. TNFα appears to play astrong role in the pathogenesis of these syndromes and heightenedconcentrations of TNFα are found in the joint, skin and gut of patientsafflicted with SpA. Etanercept has been approved for treatment ofpsoriatic arthritis. Infliximab used in two open studies demonstratedsignificant clinical benefit against SpA (Van den Bosch F., et. al. Ann.Rheum. Dis. 2000, 59:428-433; Baete D., et. al. Arhritis. Rheum. 2001,44:186-95) and psoriatic arthritis (Ogilvie A. L. et al, Br. J.Dermatol. 2001, 144:587-589). The common mechanism of action of thesedrugs and their mutual ability to improve clinical outcome suggest thatanti-TNFα therapy is useful in the treatment of SpA.

The local release of TNFα contributes to the inflammatory skin diseasepsoriasis. Concentrations of TNFα and soluble TNFα receptors (p55 andp75) were higher in lesional stratum corneum extracts of psoriaticpatients than controls (Ettehadi P., et. al. Clin. Exp. Immunol. 1994,96:146-151). These results confirm the presence of immunoreactive TNFαand quantifiable concentrations of soluble TNF receptors, which mayregulate the effects of TNFα. A number of case reports and smallclinical studies indicate that infliximab improved psoriasis symptoms(Chaudhari U., et. al. Lancet 2001, 357:1842-1847; Newland M. R., Int.J. Dermatol. 2002, 41:449-452; Schopf R. E., et. al. J. Am. Acad.Dennatol. 2002,46:886-91). Etanercept has also been clinically evaluatedand shows efficacy against psoriasis symptoms. Biologically active IL-1βhas also been found in psoriatic scales (Lundqvist E. N., et. al. Eur.J. Immunol. 1997, 27:2165-2171) suggesting a role for this cytokine inthe pathogenesis of psoriasis. Again, the interaction of variouscytokines appears to be important in this disease and suggests thatefficacious treatments would target a variety of cytokines.

Overproduction of TNFα also contributes to the clinical features ofnumerous autoimmune diseases such as diabetes. Therapies directed atTNFα have been shown to provide clinical benefit in Type II diabetes.For example, thiazolidinedione derivatives are a class of drugs thatactivate the peroxisome proliferator-activated receptor-gamma (PPARγ)resulting in diminished macrophage activation and decreased productionof TNFα (Ricote M., et. al. Nature 1998, 391:79-82; Jiang C., et. al.Nature 1998, 391:82-86). These drugs have been tested in various humanclinical studies in non-insulin-dependent diabetes and these drugsimproved control of glucose metabolism and blood lipid profiles (KumarS., et. al. Diabetologia 1996, 39:701-709; Berkowitz K., et. al.Diabetes 1996, 45:1572-1579; Yamasaki Y. et. al. Tohoku J. Exp. Med.1997, 183:173-183) in addition to lowering TNFα levels in plasma(Katsuki A., et. al. Diabete. Obes. Metab. 2000, 2:189-2191).

Systemic lupus erythematosus (SLE) is another autoimmune disorderprecipitated by increased TNFα levels. Within lupus patients, serumC-reactive protein, IL-1β and TNFα levels were higher than in controlssuggesting that an over-exuberant immune response plays a role in thedisease (Liou L. B. Clin. Exp. Rheumatol. 2001, 19:515-523). A study ofpatients with one form of SLE, neuropsychiatric lupus erythematosus(NPLE), showed that the number of peripheral blood mononuclear cellsexpressing mRNA for TNFα as well as the cerebrospinal fluid level of NOmetabolites correlated with NPLE disease severity (Svenungsson E., etal. Ann. Rheum. Dis. 2001, 60:372-9). In a study by Segal, (Segal R.,et. al. Lupus 2001, 10:23-31), SLE was induced in mice with humananti-DNA antibodies. These mice were then treated with an anti-TNFαantibody or pentoxifylline, a phosphodiesterase inhibitor that lowersTNFα production. Both treatments reduced the production of TNFα andlowered serum levels of anti-DNA antibodies. The anti-inflammatorycytokine IL-10 has been shown to regulate murine models of lupus.IL-10^(−/−) knockout mice were bred into a lupus susceptible mousestrain. These mice developed more severe lupus and suffered highermortality than IL-10^(+/+) mice (Yin Z., et. al. J. Immunol. 2002,169:2148-2155). An ideal SLE therapy would target multiple cytokines;inhibiting TNFα levels while enhancing or exerting no inhibition onIL-10 levels.

TNFα is also involved in cutaneous forms of lupus. A-308A polymorphismof the human TNFα promoter has a significantly increased prevalence inpatients suffering from subacute cutaneous LE. This polymorphism led tosubstantially higher induction of TNFα after exposure to UVB than wildtype, contributing to the photosensitivity seen in this disease (WerthV. P., et. al. J. Invest. Dermatol. 2000, 115:726-730). Biopsies frompatients with localized discoid LE showed significantly elevated levelsof IL-2 and IFNγ mRNA and elevated levels of TNFα mRNA compared tonormal skin (Toro J. R., et. al. Arch. Dernatol. 2000, 136:1497-1501).Overall, these findings suggest that anti-TNFα therapy would benefitpatients with these forms of LE.

Multiple sclerosis is an inflammatory demyelinating disease of thecentral nervous system characterized by a T-cell mediated autoimmuneresponse to the myelin sheath. A number of pro-inflammatory cytokinescontribute to the ongoing inflammation in human disease (Sharief M. K.,et. al. N. Engl. J. Med. 1991, 325:467-472) and murine experimentalautoimmune encephalomyelitis (EAE) models (Conlon P., et. al. Neurobiol.Dis. 1999, 6:149-66). Administration of anti-TNFα antibodies in the EAEanimal model reversed demyelination and paralysis (Selmaj K., et. al.Ann. Neurol. 1991, 30:694-700; Karin N., et. al. J. Exp. Med. 1994,180:2227-37). Human trials with monoclonal antibodies and soluble TNFαreceptor have been unsuccessful, emphasizing the need to develop newanti-TNFα therapies (van Oosten, B. W. et. al. Neurology1996,47:1531-1534).

TNFα has also been found to potently upregulate human immunodeficiencyvirus 1 (HIV-1) expression in T cell clones (Duh E. J., et. al. Proc.Natl. Acad. Sci. USA 1989, 86:5974-8; Folks T. M., et. al. Proc. Natl.Acad. Sci. USA 1989, 86:2365-2368; Clouse K. A., et. al. J Immunol.1989, 142:431-8) and monocytes (Koyanagi Y., et. al. Science 1988,241:1673-1675). TNFα enhances HIV-1 replication in T cells by increasingthe surface density of the HIV docking receptor, CXCR4 (Biswas P., et.al. Cytokine 2001, 13:55-59). In vivo effects of TNFα were demonstratedin a study using homozygous HIV-1 transgenic mice. These mice havesignificantly increased serum TNFα levels and die within 3-4 weeks.Treatment with an antibody to TNFα prevented death, decreasedcharacteristic skin lesions in these mice, and profoundly reduced HIV-1expression (De S. K., et. al. J. Virol. 2002, 76:11710-4). Thus,inhibition of TNFα could suppress T cell activation, CXCR4 expressionand slow viral spread and replication.

Pro-inflammatory cytokines have been implicated in other viralinfections including the cytomegalovirus, influenza virus and the herpesfamily of viruses. TNFα enhances the basal activity of the majorimmediate early enhancer/promoter of human cytomegalovirus and may playa role in reactivation of latent HCMV infection in premonocytic cells(Prosch S., et. al. Virology 1995, 208:197-206). Likewise, GM-CSFenhanced de novo influenza A virus protein synthesis, viral particlerelease and cell death in human monocytes infected by influenza A virus.Treatment of persistently herpes simplex virus (HSV) infectedmacrophages for 2 weeks with TNFα resulted in an increase of HSV yieldand an increase in virus-induced cytotoxic effects (Domke-Opitz I., et.al. Scand. J. Immunol. 1990, 32:69-75). Further studies showed that TNFαand possibly GM-CSF enhanced the reactivation frequency and replicationof HSV in the trigeminal ganglia of mice latently infected with HSV(Walev I., et. al. Arch. Virol. 1995, 140:987-992).

A number of cytokines contribute to the demise of patients sufferingfrom sepsis or endotoxic shock. TNFα and IL-1β have a well-establishedcentral role in sepsis, septic shock and endotoxic shock. Increasedlevels of these cytokines are associated with fever, hypotension andshock (Smith J. W. et. al. J. Clin. Oncol. 1992, 10:1141-1152; ChapmanP. B., et. al. J. Clin. Oncol. 1987, 5:1942-1951) together with theinduction of gene expression for phospholipase A2 (Gronich J., et. al.J. Clin. Invest. 1994, 93:1224-1233) and NO synthase. The induction ofNO from smooth muscle cells mediates decreased mean arterial pressureand systemic vascular resistance during septic shock, suggesting afundamental role for NO. Therapies targeting TNFα in particular withdownregulatory effects on IL-1β and NO could be beneficial in thetreatment of sepsis, septic shock, and endotoxic shock.

A variety of cell types are involved in the inflammatory process.Overproduction of TNFα by monocytes, macrophages and other immune cellsis a key element in the pathogenesis of a multitude of diseases.Macrophages and T-cells in particular play a central role in theinitiation and maintenance of the immune response. Once activated bypathological or immunogenic stimuli, macrophages respond by releasing ahost of cytokines, including TNFα, IL-1β, IL-8, IL-12, NO, IL-6, GM-CSF,G-CSF, M-CSF and others. T-cells release IL-2, IL-4, interferon-γ, andother inflammatory cytokines. These cytokines activate other immunecells and some can also act as cytotoxic agents alone. Excessive releaseof macrophage and T-cell derived inflammatory mediators can thereforelead to damage of normal cells and surrounding tissues. Theoverabundance of these cytokines is a clinical feature of many chronicinflammatory diseases. Treatment and resolution of these conditions maydepend on attenuation of the immune cells, particularly the macrophagesand T-cells.

Previous reports suggest that polyamines modulate macrophage function byinhibiting the secretion of inflammatory mediators such as TNFα (ZhangM., et. al. J. Exp. Med. 1997, 185, 1759-68). Treatment of humanperipheral blood mononuclear cells or a murine macrophage cell line withspermine prior to stimulation with the immunostimulantlipopolysaccharide (LPS) inhibited the release of pro-inflammatorycytokines including TNFα. Spermine treatment had no effect on increasesin TNFα mRNA levels, indicating that the suppression occurredpost-transcriptionally. In subsequent studies, Zhang et al reported thatpretreatment of cells with a polyamine transport inhibitor prior toaddition of spermine and LPS restored TNFα levels to those seen in theabsence of spermine (Zhang M., et. al. Mol. Med. 1999, 5:595-605). Theinhibitory action of spermine may provide a natural mechanism forattenuation of the immune response and protection against excessiveinflammatory damage. Furthermore, a U.S. patent (Bergeron, R. J. U.S.Pat. No. 5,843,959) describes bicyclic polyamine compositions thatexerted anti-inflammatory effects. These compositions provided modestinhibition of type II collagen-induced arthritis in mice andcarrageenan-induced edema in rat paws. An additional U.S. patent(Tracey, K. J. et. al. U.S. Pat. No. 6,482,833) describes spermineantagonists that prevent spermine-induced immunosuppression. Thus theutility of polyamine analog-based therapies in treating inflammatoryconditions has been established.

SUMMARY OF THE INVENTION

The present invention is directed to novel polyamine analogs andderivatives and methods for their use in the inhibition of theexpression or activity of TNFα and/or one or more other cytokinesincluding but not limited to IL-1β, IL-2, IL-6, NO, GM-CSF,interferon-γ(IFN-γ), G-CSF, M-CSF, IL-8, IL-12, and IL-18. The analogand derivative compounds of the invention have the ability to reducedetectable levels of TNFα produced or secreted by cells under conditionswhere TNFα production or secretion would occur. The compounds also mayor may not have effects on other pro- or anti-inflammatory cytokines.Without being bound by theory, it is believed that the compounds of theinvention inhibit the production or secretion of TNFα from cells underconditions where TNFα production or secretion would occur. The compoundsof the invention may also inhibit the production or secretion of one ormore other cytokines from cells under conditions where cytokineproduction or secretion would occur. They may be used independently orin combination with any other agent that exerts an effect againstinflammatory, infectious, autoimmune or other proliferative diseases andconditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of preferred compounds, MQT 100 and MQT 600.

FIG. 2 shows the reaction scheme for the synthesis of analogs of MQT 100(Series 100 and 200). Reagents and conditions: a) Boc₂O, Na₂CO₃,THF/H₂O; b) 2-nitrobenzenesulfonyl chloride, CH₂Cl₂, Et₃N; c)N-phthalimido-3-amino-1-propanol, Ph₃P, DIAD, THF; d) NH₂NH₂, EtOH,reflux; e) N-Boc-6-amino-1-hexanol, Ph₃P, DIAD, THF; f) HSCH₂CH₂OH, DBU,DMF; g) 3N HCl in MeOH.

FIG. 3 is a tabular representation of the conformational analysis ofSeries 200 analogs.

FIG. 4 shows the reaction scheme for the synthesis of “amino-walk”analogs. Reagents and conditions: Use of intermediate A is shown as anexample in this route. a) Phthalic anhydride, EtOH, reflux; b)HOCH₂(CH₂)_(b)CH₂NPhth, Ph₃P, DIAD, THF, b=0 to 12; c) NH₂NH₂, EtOH,reflux; d) HOCH₂(CH₂)_(n)CH₂NPhth, Ph₃P, DIAD, THF, c=0 to 12; e)2-nitrobenzenesulfonyl chloride, CH₂Cl₂, Et₃N; f) HSCH₂CH₂OH, DBU, DMF;g) 3N HCl in MeOH.

FIG. 5 is a tabular representation of representative series 300 and 400“amino-walk” analogs.

FIG. 6 shows representative examples of methylated “metabolism-proof”molecules.

FIG. 7 shows the reaction scheme for the synthesis of series 600 andseries 700 (truncated) molecules. Reagents and conditions: a)HOCH₂(CH₂)_(b)CH₂NPhth, Ph₃P, DIAD, THF, b=0 to 12; b) NH₂NH₂, EtOH,reflux; c) 2-nitrobenzenesulfonyl chloride, CH₂Cl₂, Et₃N; d)HOCH₂(CH₂)_(c)CHRNPhth, Ph₃P, DIAD, THF, c=0 to 12; e) HSCH₂CH₂OH, DBU,DMF; f) 3N HCl in MeOH.

FIG. 8 shows the reaction scheme for the synthesis of aromaticsubstituted amino alcohol precursor molecules. Reagents and conditions:a) methyl 2-((succinimidooxy)carbonyl)benzoate, CH₃CN, H₂O, Na₂CO₃; b)i. Isobutylchloroformate, Et₃N, THF ii. NaBH₄, MeOH.

FIG. 9 is a tabular representation of precursors and examples of series600 molecules.

FIG. 10 is a tabular representation of series 700 analogs.

FIG. 11 shows the structure of two representative combination analogs(Series 800).

FIG. 12 shows the reaction scheme for the enzymatic resolution of MQT600 stereoisomeric precursor mixture. Reagents and conditions: a)NH₄OAc, CH₂(CO₂H)₂, EtOH, reflux; b) H₂SO₄, EtOH, reflux; c) AmanoPS,H₂O.

FIG. 13 shows the reaction scheme for the synthesis of MQT 600enantiopure precursors via chemical synthesis from stereopureprecursors. Reagents and conditions: a) i. ^(i)BuOCOCl,N-methylmorpholine, THF, ii. CH₂N₂, ether; iii. CF₃CO₂Ag,N-methylmorpholine, H₂O, THF.

FIG. 14 is a tabular representation of the Analog Summary.

FIG. 15 shows the results from analysis of MQT 100 and MQT 600 for cellgrowth inhibition (referred to as IC₅₀) and inhibition of LPS-inducedTNFα release into the extracellular environment (referred to as EC₅₀).

BEST AND VARIOUS MODES FOR CARRYING OUT INVENTION

The compounds and derivatives of the invention include those encompassedby the following formula I:

wherein, a, b and c may be the same or different and are integers from 0to 12, X equals NH or CHNH₂, R₁ and R₂ can be the same or different andequal to a hydrogen or a straight or branched C₁ to C₂₀ saturated orunsaturated aliphatic such as methyl; aliphatic amine; an alicyclic;single or multi-ring aromatic such as 1- or 2-naphthylene; a single ormulti-ring aromatic heterocycle; a single or multi-ring saturatedheterocycle and the halogenated forms thereof.

The compounds and derivatives of the invention also include thoseencompassed by the following formula II:

wherein, a, b and c may be the same or different and are integers from 0to 12; R₁, R₂, R₃, and R₄ can be the same or different and equal to ahydrogen or a straight or branched C₁ to C₂₀ saturated or unsaturatedaliphatic such as methyl; aliphatic amine; an alicyclic; single ormulti-ring aromatic such as 1- or 2-naphthylene; a single or multi-ringaromatic heterocycle; a single or multi-ring saturated heterocycle andthe halogenated forms thereof.

A preferred aspect of the invention relates to a compound or derivativethat is a potent inhibitor of inflammatory, infectious, autoimmune orother proliferative diseases and conditions by reducing or inhibitingthe expression or secretion of TNFα outside the cell and/or modulatingthe expression or secretion of one or more other involved cytokines.

The present invention also provides for the free base or acid forms, aswell as salts thereof, of the polyamine analogs and derivativesdescribed herein. The invention also includes the optical isomers of thedisclosed analogs and derivatives. In a further aspect of the invention,mixtures of enantiomers and/or diastereoisomers, resulting from a singlepreparative step, combination, or interconversion are encompassed.

The invention also provides for the prodrug forms of the above describedanalogs and derivatives, wherein the prodrug is metabolized in vivo toproduce an analog or derivative as set forth above. Indeed, some of theabove described analogs or derivatives may be a prodrug for anotheranalog or derivative.

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

The term “aryl” refers to monocyclic or multiring aromatic hydrocarbongroups typically containing 6 to 14 carbon atoms in the ring portion,such as phenyl, naphthyl, biphenyl and diphenyl groups, each of whichmay be substituted.

The term “saturated aliphatic” refers to straight or branched chainunsubstituted hydrocarbon groups typically having 1 to 20 carbon atoms,more typically 1 to 8 carbon atoms. The expression “lower alkyl” refersto unsubstituted alkyl groups of 1 to 4 carbon atoms.

Examples of suitable saturated aliphatic or alkyl groups include methyl,ethyl and propyl. Examples of branched alkyl groups include isopropyland t-butyl.

The term “halogen” or “halo” refers to fluorine, chlorine, bromine andiodine.

Examples of multiring aromatic (unsaturated) heterocycle groups are2-quinolinyl, 3-quinolinyl, 5-quinolinyl, 6-quinolinyl, 7-quinolinyl,1-isoquinolinyl, 3-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl,3-cinnolyl, 6-cinnolyl, 7-cinnolyl, 2-quinazolinyl, 4-quinazolinyl,6-quinazolinyl, 7-quinazolinyl, 2-quinoxalinyl, 5-quinoxalinyl,6-quinoxalinyl, 1-phthalaonyl, 6-phthalazinyl, 1-5-naphthyridin-2-yl,1,5-naphthyridin-3-yl, 1,6-naphthyridin-3-yl, 1,6-naphthyridin-7-yl,1,7-naphthyridin-3-yl, 1,7-naphth7yridin-6-yl, 1,8-naphthyrdiin-3-yl,2,6-naphthyridin-6-yl, 2,7-naphthyridin-3-yl, indolyl, 1H-indazolyl,purinyl and pteridinyl.

Examples of single ring heterocycle groups are pyrrolyl, pyranyl,oxazolyl, thiazoyl, thiophenyl, furanyl, imidazolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, isothiazolyl andisoxazolyl.

Examples of saturated heterocycle groups are pyrrolidinyl,imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, andmorpholinyl.

The heterocycle groups contain N, O and/or S and typically contain 5 to10 atoms in the ring(s).

Examples of suitable alicyclic groups are cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

Examples of suitable aliphatic amine groups are methylamine, ethylamine,propylamine, isopropylamine, tert-butyl amine and diethylamine. Theamine groups include diamines, and triamines and can be primary,secondary or tertiary amines.

Typically the amines 1-20 contain carbon atoms and more typically 1-8carbon atoms.

The compounds of the present invention can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

Prodrug forms of the compounds bearing various nitrogen functions(amino, hydroxyamino, hydrazino, guanidino, amidino, amide, etc.) mayinclude the following types of derivatives where each R groupindividually may be hydrogen, substituted or unsubstituted alkyl, aryl,alkenyl, alkynyl, heterocycle, alkylaryl, aralkyl, aralkenyl, aralkynyl,cycloalkyl or cycloalkenyl groups as defined above.

Carboxamides, —NHC(O)R

Carbamates, —NHC(O)OR

(Acyloxy)alkyl Carbamates, NHC(O)OROC(O)R

Enamines, —NHCR(═CHCRO₂R) or —NHCR(═CHCRONR₂)

Schiff Bases, —N═CR₂

Mannich Bases (from carboximide compounds), RCONHCH₂NR₂

Preparations of such prodrug derivatives are discussed in variousliterature sources (examples are: Alexander et al., J. Med. Chem. 1988,31, 318; Aligas-Martin et al., PCT WO pp/41531, p. 30). The nitrogenfunction converted in preparing these derivatives is one (or more) ofthe nitrogen atoms of a compound of the invention.

Prodrug forms of carboxyl-bearing compounds of the invention includeesters (—CO₂R) where the R group corresponds to any alcohol whoserelease in the body through enzymatic or hydrolytic processes would beat pharmaceutically acceptable levels.

Another prodrug derived from a carboxylic acid form of the invention maybe a quaternary salt type

of structure described by Bodor et al., J. Med. Chem. 1980, 23, 469.

It is of course understood that the compounds of the present inventionrelate to all optical isomers and stereo-isomers at the various possibleatoms of the molecule.

The compounds of this invention form acid and base addition salts with awide variety of organic and inorganic acids and bases and includes thephysiologically acceptable salts which are often used in pharmaceuticalchemistry. Such salts are also part of this invention. Typical inorganicacids used to form such salts include hydrochloric, hydrobromic,hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like.Salts derived from organic acids, such as aliphatic mono anddicarboxylic acids, phenyl substituted alkonic acids, hydroxyalkanoicand hydroxyalkandioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, may also be used. Such pharmaceutically acceptable saltsthus include acetate, phenylacetate, trifluoroacetate, acrylate,ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate,methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, cabrate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzene-sulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toleunesulfonate,xylenesulfonate, tartarate, and the like.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates, as well asaliphatic and primary, secondary and tertiary amines, aliphaticdiamines. Bases especially useful in the preparation of addition saltsinclude sodium hydroxide, potassium hydroxide, ammonium hydroxide,potassium carbonate, methylamine, diethylamine, and ethylene diamine.

The compounds may be utilized alone or in combination with other agents,particularly other inhibitors of polyamine synthesis or transport, butincluding other inhibitors of cell proliferation.

In another aspect of the invention, compositions containing the abovedescribed compounds and derivatives are provided. Preferably, thecompositions are formulated to be suitable for pharmaceutical oragricultural use by the inclusion of appropriate carriers or excipientsand/or anti-inflammatory agents.

In another aspect of the invention, compositions containing thedisclosed analogs and derivatives are provided. Preferably, thecompositions are formulated to be suitable for pharmaceutical use by theinclusion of one or more appropriate carriers or excipients.

In a further aspect of the invention, methods for the use of the abovedescribed analogs and derivatives, as well as compositions, areprovided. These methods include uses of the invention's compounds toinhibit, retard or modulate the production, secretion, expression and/oractivity of TNFα and/or one or more other involved cytokines to treatconditions associated with the unwanted or undesirable presence oractivity of TNFα and/or one or more other involved cytokines. Examplesof human diseases and conditions include, but are not limited to,chronic or acute inflammation, inflammatory bowel disease (includingCrohn's disease), inflammatory bowel syndrome, autoimmune diseases,rheumatoid arthritis, systemic lupus erythematosus, cutaneous forms oflupus, diabetes, multiple sclerosis, psoriasis, spondyloarthropathies(SpA) including spondylitis, synovitis, psoriatic arthritis andsubclinical gut inflammation and infectious diseases including sepsis,septic shock, endotoxic shock, HIV and other viral infections includingcytomegalovirus, herpes simplex virus, influenza virus and otherproliferative diseases and disorders including but not limited tocancer.

A preferred compound according to the present invention is designated asMQT 100 which is shown in FIG. 1. This compound inhibited TNFαproduction in a reproducible and robust manner. Another preferredcompound is shown in FIG. 1 and designated as MQT 600.

The potent molecule MQT 100 as well as analogs thereof (e.g., series 100and 200) can be synthesized via the route shown in FIG. 2. The syntheticroute began by taking the six possible different isomers ofdiaminocyclohexane and mono-protecting them as theirmono-^(t)butoxycarbonyl carbamates followed by derivatization with a2-nitrobenzenesulfonyl group (Fukuyama, T., et. al. Tetrahedron Lett.1997, 38, 5831-5834) to give intermediates A-F. These intermediates werethen each individually coupled to N-(3-hydroxypropyl)phthalimide via aMitsunobu reaction to give orthogonally protected intermediates G-L.Compounds G-L were used to produce two different series of finalproducts. In one case, they were simply deprotected to give the sixtruncated analogs in SERIES 200. In the other series, G-L were subjectedto another round of Mitsunobu extension with N-Boc-6-amino-1-hexanol togive analogs in SERIES 100 following their deprotection.

This series was tested for bioactivity and the results can be related tothe arrangement of the two amino groups attached to the cyclohexanering. Conformational analysis of this ring arrangement shows that theenergy-minimized (performed using CambridgeSoft Chem3D version 5.0)structures have dramatically different spacing of these two groups. FIG.3 shows the resulting structures for Series 200 together with thedesignations given them for convenience. The distance between these twoamino groups as measured in the minimized structure are shown in FIG. 3.

The series 300 and 400 compounds are exemplary of compounds of thepresent invention wherein the distance between the nitrogen atoms in thelinear portion of the molecule is varied (“nitrogen-scan” or“amino-walk” analogs). Modification of the distance between the fournitrogen atoms, and thus the number of methylene units between them, iscarried out by a variation on the synthetic scheme shown in FIG. 2. Thismodified route is shown in FIG. 4. The N-phthalimido protected aminoalcohols were produced via standard conditions by refluxing the aminoalcohols overnight with phthalic anhydride in ethanol. The resulting setof precursors was utilized in the route shown to give the analogsdescribed in FIG. 5. Variations in the geometric and stereochemicalarrangement of the diamine groups of the cyclohexane portion of themolecule are also envisioned via the use of the intermediates A-F shownin FIG. 2, as precursors in the scheme in FIG. 4.

Additional analogs bearing various branched or substitutedfunctionalities can be readily envisioned. These modified analogs aretailored to possess features that increase their usefulness inbiological systems. As an example, the addition of a methyl group to thecarbon atom next to a nitrogen atom, especially at an aminopropylportion, greatly reduces its ability to be metabolized by a variety ofpolyamine and amino oxidase enzymes. An analog of the preferredcompounds addressing this modification is shown in FIG. 6.

A variety of aromatic substituted amino alcohol precursor molecules wereutilized in the scheme shown in FIG. 7 to provide bioactive compoundshaving the naphthylene ring moiety processed by MQT 600.

The aromatic substituted amino alcohol precursors were produced via theroute shown in FIG. 8. Use of a new and efficient reagent enabled theN-phthaloylation of a variety of amino acid precursors (Casimir J. R.,et. al. J. Org. Chem. 2002, 67, 3764-3768). The resulting N-phthalimideprotected a and β-amino acids were then converted to their isobutylmixed anhydrides and reduced in situ with NaBH₄ (Rodriquez, et. al.Tetrahedron Lett. 1991, 32, 923-926). These protected amino alcoholswere used in the FIG. 7 route. FIG. 9 shows examples of the moleculesproduced in the route shown in FIG. 7 (SERIES 600 analogs). FIG. 10shows examples of truncated molecules also produced by the route shownin FIG. 7 (SERIES 700 analogs). Several of these examples alsoincorporate analogs produced by changing the position of the aminogroups along the polyamine backbone (the “amino-walk” analogs).Variations in the number of methylene (—CH₂—) groups between the aminofunctions not only influence the affinity for the biological target butalso have a dramatic effect on the physical properties of the drugmolecule. An example of a property affected includes the hydrophobicnature of the molecule. This will greatly influence the molecule'sability to penetrate biological membranes to ultimately interact withits biological target. It will also greatly impact the molecule's routeof administration in the clinical setting. The most desirable featurescan be tailored into the molecule by adjusting the structure in acontrolled and thoughtful manner once aware of the present disclosure. Afield of pharmaceutical development called ADME (absorption,distribution, metabolism and elimination) has evolved to address thesefeatures of drug characteristics.

A variety of molecules can be envisioned that combine the optimizedfunctionalities in the described series. Examples of combinationmolecules are shown in FIG. 11, SERIES 800.

A variety of compounds described in this invention can exist inalternate stereochemical forms and various chemical methodologies existfor the separation of one enantiomeric form from the other. As examples,MQT 600 can be separated into its enantiomeric components via one ofthree different methods. The first and most straightforward method wouldbe the chromatographic or crystallographic resolution of the mixture.These methods are well established in the field and give the advantagethat the two isomers can be obtained in pure form for testing. On theother hand, resolution via these methods by theory reduces the yield ofthe isomer of interest to <50% at best. Also, resolution by thesemethods still requires determination of the absolute stereochemicalassignment of the individual isomers. For these reasons it is sometimesbetter to produce the stereoisomers in their pure form through synthesisfrom stereochemically pure precursors whereby the absolute form has beendefined.

A second approach to synthesize the required stereoisomeric pair of MQT600 isomers would entail an enzymatic resolution step. FIG. 12 shows aroute by which a racemic mixture of β-aminoacid esters is produced via aRadionow reaction (Rodionow, W. M., et. al. J. Am. Chem. Soc. 1929, 51,841-847) followed by acid-catalyzed esterification. This mixture is thentreated with the esterase enzyme Amano PS that has been shown toselectively hydrolyze the (S)-isomer ester (Faulconbridge, S. J. et. al.Tetrahedron Lett, 2000, 41, 2679-2681). The resulting resolvedester/acid forms can be converted into the desired MQT 600 R- andS-isomers using the routes shown in this application.

Alternatively, the stereo-pure components of MQT 600 can be obtained bypurchasing their stereo-defined forms and using them in the syntheticroutes outlined in this application. The R- or S-enantiopure β-aminoacids 1-naphthyleneglycine in their FMOC protected forms are availablefrom BaChem AG (Switzerland). These can be homologated via anArndt-Eistert reaction to give the FMOC-β-amino acids (Guichard, G. et.al. Helv. Chim. Acta 1998, 81, 187) shown in FIG. 13. These isomers canthen be transformed to precursors used in the ultimate production of theisomeric forms of MQT 600 by methodology outlined in this application.

A summary of the different analogs envisioned by this invention is shownin FIG. 14.

The following non-limiting examples are presented to further illustratean understanding of the present invention.

EXAMPLE 1 Synthesis of MOT 100

All chemical reagents and starting materials were of the highest gradeavailable and were used without further purification. Thin-layerchromatography analysis of crude reaction products and columnchromatography was performed using Merck F₂₅₄ silica gel plates andBaker 40 μm flash chromatography packing, respectively.

Trans-N-Boc-4-aminocyclohexanol—A 1.52 g (10 mmol) portion oftrans-4-aminocyclohexanol hydrochloride was dissolved in 10 mL of 10%aqueous sodium carbonate and 5 mL THF and stirred at room temp. Asolution of Boc₂O in 15 mL THF was added and the solution stirred for 18hours at room temp. The resulting solution was concentrated in vacuo andthen partitioned between 50 mL H₂O and 50 mL EtOAc. The aqueous layerwas once more extracted with EtOAc and the combined organic extractswere washed with 1N HCl and brine. The organic extract was dried overMgSO₄ and evaporated in vacuo to give 2.02 g of a white solid. (94%).R_(f) 0.26 (1:1 Hexane:EtOAc). This material was used directly in thenext step without purification.

Trans-N-Boc-4-amino-1-phthalimido-cyclohexane—A solution of 1.5 g (7.0mmol) of trans-N-Boc-4-aminocylcohexanol, 1.15 g (7.8 mmol) ofphthalimide and 2.0 g (7.6 mmol) of Ph₃P dissolved in 50 mL anhydrousTHF was stirred at 0° C. under argon. A neat aliquiot of DIAD (1.67 mL,8.5 mmol) was then added dropwise. The yellow solution was stirred for18 hours at 25° C. at which time it was concentrated in vacuo. Theproduct was purified by silica gel chromatography eluting first with 8:2Hexane/EtOAc, then with 1:1 Hexane/EtOAc. A yield of 500 mg of clear oil(21%) was obtained following evaporation of product fractions asdetermined by TLC with ninhydrin detection (R_(f) 0.68 (1:1Hexane:EtOAc)).

N⁴-Boc-N¹-Nbs-trans-1,4-diaminocyclohexane—A solution of 500 mg (1.5mmol) of trans-N-Boc-4-amino-1-phthalimido-cyclohexane in 20 mL EtOH wastreated with 151 μl of hydrazine hydrate. The resulting solution wasstirred at room temp for 18 hours at which time the precipitate that hadformed was filtered off. The filtrate was evaporated to dryness to givea total of 422 mg of the mono-Boc-1,4-diamino intermediate. Thiscompound was then dissolved in 15 mL CH₂Cl₂ and 554 mg (2.5 mmol)2-nitrobenzenesulfonyl chloride was added and the resulting solution wasstirred under argon at 0° C. A portion of 348 μL of triethylamine (2.5mmol) was then added and the solution was stirred at 25° C. for 2 hours.The reaction was then poured into aqueous 0.1N HCl and then immediatelyextracted with 3×50 mL portions of CH₂Cl₂. The combined organic extractswere washed with brine, dried over MgSO₄ and then evaporated to dryness.The resulting crude white solid was purified on silica gel eluting with6:4 Hexane/EtOAc to give 415 mg (53% yield) of clear oil product (R_(f)0.40 (1:1 Hexane/EtOAc)).

N¹-Boc-N⁴-Nbs-N⁴-(3-propyl-1-phthalimide)-trans-1,4-diaminocyclohexane—Asolution of 310 mg (0.78 mmol) ofN⁴-Boc-N¹-Nbs—trans-1,4-diaminocyclohexane was dissolved in 20 mL of 1:3CH₂Cl₂/benzene together with 254 mg (0.97 mmol) of Ph₃P and 127 mg (0.62mmol) of N-(3-hydroxypropyl)phthalimide. The resulting solution wasstirred at 25° C. under argon. To this solution was added 191 μL (0.97mmol) of a 40% solution of DIAD in toluene dropwise. After addition wascomplete, stirring was continued for an additional 18 hours. Thesolution was evaporated to dryness and the crude oil purified on silicagel eluting first with 8:2 Hexane/EtOAc then with 1:1 Hexane/EtOAc togive a total of 322 mg (89%) of product as a clear oil (R_(f) 0.35 (1:1Hexane/EtOAc)).

N¹-Boc-N⁴-Nbs-N⁴-(3-propyl-1-Nbs-amino)-trans-1,4-diaminocyclohexane—Toa solution of 322 mg (0.55 mmol) ofN-Boc-N⁴-Nbs-N⁴-(3-propyl-1-phthalimide)-trans-1,4-diaminocyclohexane in10 mL of absolute EtOH was added 50 μL of neat hydrazine hydrate. Thesolution was stirred at room temp for 18 hours at which time the whiteprecipitate was filtered off. Following TLC analysis of the filtrate toshow no starting material remained, the solvent was evaporated to give150 mg of white solid. This was taken immediately to next step bydissolving in 10 mL CH₂Cl₂ together with 93 mg (0.42 mmol) of2-nitrobenzenesulfonyl chloride and stirring under argon at 0° C. A 59μL (0.42 mmol) portion of dry triethylamine was then added and thesolution was allowed to warm to 25° C. and stirred for an additional 18hours. The solution was then poured into aqueous 0.1 N HCl andimmediately extracted 3×25 mL CH₂Cl₂. The combined organic extracts werewashed with brine, dried over MgSO₄ and evaporated to dryness. The crudewhite solid was purified on silica gel eluting with 1:1 Hexane/EtOAc togive 142 mg (40% yield) of product as a pale yellow oil (R_(f) 0.50(48:48:4 Hexane/EtOAc/MeOH)).

N¹-Boc-N⁴-Nbs-N⁴-(3-propyl-1-Nbs-amino-1-(6-hexanyl-1-Boc-amino))-trans-1,4-diaminocyclohexaneTo a solution of 56 mg (0.087 mmol) ofN¹-Boc-N⁴-Nbs-N⁴-(3-propyl-1-Nbs-amino)-trans-1,4-diaminocyclohexanedissolved in 3.0 mL of 1:3 CH₂Cl₂/benzene together with 30 mg (0.11mmol) of Ph₃P and 17 mg (0.080 mmol) of N⁶-Boc-aminohexanol at 25° C.under argon was added 22 μL (0.11 mmol) of a 40% solution of DIAD intoluene. After addition was complete the reaction was stirred for anadditional 18 hrs. The solution was evaporated to dryness and the crudeoil was then purified on silica gel, eluting first with 8:2Hexane/EtOAc, then with 1:1 Hexane/EtOAc, and finally with 48:48:4Hexane/EtOAc/MeOH to give 53 mg (79%) of product as a clear oil (R_(f)0.30 (48:48:4 Hexane/EtOAc/MeOH)).

MQT 100—To a solution of 53 mg (0.063 mmol) ofN¹-Boc-N⁴-Nbs-N⁴-(3-propyl-1-Nbs-amino-1-(6-hexanyl-1-Boc-amino))-trans-1,4-diaminocyclohexanein 2 mL of THF was added 250 μL of DBU together with 50 μL of2-mercaptoethanol. The resulting solution was stirred at 25° C. for 18hours and then evaporated under a stream of argon. The resulting productwas purified on silica gel eluting first with 90:10 CHCl₃/MeOH then with85:14:1 CHCl₃/MeOH/NH₄OH and finally with 80:18:2 CHCl₃/MeOH/NH₄OH togive 25 mg of the di-Boc-intermediate (R_(f) 0.26, 80:18:2CHCl₃/MeOH/NH₄OH). This product was then dissolved in 2 mL of 3N HCl inMeOH and stirred at 25° C. for 18 hours. The solution was evaporated todryness, redissolved in a small volume of water and then evaporated todryness again to give 12 mg (46%) of the HCl salt of MQT 100 as a whitesolid (R_(f) 0.16 (1:1 CH₃CN/NH₄OH)).

EXAMPLE 2 Cell Culture and Reagents

The RAW264.7 cell line was obtained from ATCC (Manassas, Va.) andcultured in the recommended media, serum, and CO₂ concentration. Mediaswere obtained from Mediatech, Inc. (Herndon, Wash.) and serums fromGibco BRL (Gaithersburg, Md.). 50 U/mL penicillin, 50 mg/mL streptomycinand 2 mM L-glutamine (all from BioWhittaker, Walkersville, Md.) wereincluded in all cultures. When cells were cultured with compounds, 1 mMaminoguanidine (AG; Sigma) was included to inhibit serum amine oxidaseactivity.

EXAMPLE 3 RAW264.7 Growth Inhibition Assay

Compounds were screened for cell growth inhibition by exposing RAW264.7mouse macrophages to a range of compound concentrations up to 100 μM for3 days. On the fourth day, MTS/PMS dye (Cell Titer 96 AqueousNon-Radioactive Cell Proliferation Assay: Promega, Madison, Wis., USA)solution was added and the OD₄₉₀ was measured. The percent inhibition ofgrowth compared to untreated control was ascertained and the IC₅₀(concentration at which 50% of cell growth was inhibited) was computed.The numbers shown are representative of 3 independent experiments (FIG.15).

EXAMPLE 4 TNFα Inhibition

For TNFα inhibition experiments, RAW264.7 mouse macrophages were exposedto a range of concentrations of compound for 3 days. On the fourth day,an immunostimulant, LPS(Sigma, St. Louis, Mo., USA), was added to thecell medium for four hours to induce production of TNFα, followed byharvesting of the extracellular medium. TNFα levels in the supernatantwere analyzed by ELISA (R&D Systems and Endogen). The percent TNFαinhibition compared to LPS-only treated control was ascertained and theEC₅₀ (concentration at which 50% of TNFα secretion was inhibited) wascomputed. Results are the average±stdev of at least 3 independentexperiments (FIG. 15).

The pharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, or diluents, are well-known to thosewho are skilled in the art. Typically, the pharmaceutically acceptablecarrier is chemically inert to the active compounds and has nodetrimental side effects or toxicity under the conditions of use. Thepharmaceutically acceptable carriers can include polymers and polymermatrices.

The compounds of this invention can be administered by any conventionalmethod available for use in conjunction with pharmaceuticals, either asindividual therapeutic agents or in a combination of therapeutic agents.

The dosage administered will, of course, vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent and its mode and route of administration; the age, health andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; and the effectdesired. A daily dosage of active ingredient can be expected to be about0.001 to 1000 milligrams (mg) per kilogram (kg) of body weight, with thepreferred dose being 0.1 to about 30 mg/kg.

Dosage forms (compositions suitable for administration) contain fromabout 1 mg to about 500 mg of active ingredient per unit. In thesepharmaceutical compositions, the active ingredient will ordinarily bepresent in an amount of about 0.5-95% weight based on the total weightof the composition.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, and powders, or in liquid dosage forms, suchas elixirs, syrups and suspensions. It can also be administeredparenterally, in sterile liquid dosage forms. The active ingredient canalso be administered intranasally (nose drops) or by inhalation of adrug powder mist. Other dosage forms are potentially possible such asadministration transdermally, via patch mechanism or ointment. Theactive ingredient can be administered employing a sustained or delayedrelease delivery system or an immediate release delivery system.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, propylene glycol, glycerin, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant, suspending agent, or emulsifyingagent. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and corn starch.Tablet forms can include one or more of the following: lactose, sucrose,mannitol, corn starch, potato starch, alginic acid, microcrystallinecellulose, acacia, gelatin, guar gum, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, calcium stearate, zincstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acadia, emulsions, and gels containing, inaddition to the active ingredient, such carriers as are known in theart.

The compounds of the present invention, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, and nitrogen. They also may beformulated as pharmaceuticals for non-pressured preparations, such as ina nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol suchas poly(ethyleneglycol) 400, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, afatty acid ester or glyceride, or an acetylated fatty acid glyceridewith or without the addition of a pharmaceutically acceptablesurfactant, such as a soap or a detergent, suspending agent, such aspectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyldialkylammoniumhalides, and alkylpyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl β-aminopropionates, and 2-alkylimidazolinequaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically contain from about 0.5% to about25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5% toabout 15% by weight. Suitable surfactants include polyethylene sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol.

Pharmaceutically acceptable excipients are also well-known to those whoare skilled in the art. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical composition of thepresent invention. The following methods and excipients are merelyexemplary and are in no way limiting. The pharmaceutically acceptableexcipients preferably do not interfere with the action of the activeingredients and do not cause adverse side-effects. Suitable carriers andexcipients include solvents such as water, alcohol, and propyleneglycol, solid absorbants and diluents, surface active agents, suspendingagent, tableting binders, lubricants, flavors, and coloring agents.

The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.The requirements for effective pharmaceutical carriers for injectablecompositions are well known to those of ordinary skill in the art. SeePharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia,Pa., Banker and Chalmers, Eds., 238-250 (1982) and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., 622-630 (1986).

Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier; aswell as creams, emulsions, and gels containing, in addition to theactive ingredient, such carriers as are known in the art.

Additionally, formulations suitable for rectal administration may bepresented as suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases. Formulations suitable forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams, or spray formulas containing, in addition to theactive ingredient, such carriers as are known in the art to beappropriate.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the animal over a reasonable time frame. One skilled in theart will recognize that dosage will depend upon a variety of factorsincluding a condition of the animal, the body weight of the animal, aswell as the condition being treated.

A suitable dose is that which will result in a concentration of theactive agent in a patient which is known to effect the desired response.

The size of the dose also will be determined by the route, timing andfrequency of administration as well as the existence, nature, and extentof any adverse side effects that might accompany the administration ofthe compound and the desired physiological effect.

Useful pharmaceutical dosage forms for administration of the compoundsaccording to the present invention can be illustrated as follows:

Hard Shell Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatine capsules each with 100 mg of powdered activeingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesiumstearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil is prepared and injected by means of apositive displacement pump into molten gelatin to form soft gelatincapsules containing 100 mg of the active ingredient. The capsules arewashed and dried. The active ingredient can be dissolved in a mixture ofpolyethylene glycol, glycerin and sorbitol to prepare a water misciblemedicine mix.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit is 100 mg of active ingredient, 0.2 mg of colloidalsilicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystallinecellulose, 11 mg of starch, and 98.8 mg of lactose. Appropriate aqueousand non-aqueous coatings may be applied to increase palatability,improve elegance and stability or delay absorption.

Immediate Release Tablets/Capsules

These are solid oral dosage forms made by conventional and novelprocesses. These units are taken orally without water for immediatedissolution and delivery of the medication. The active ingredient ismixed in a liquid containing ingredients such as sugar, gelatin, pectinand sweeteners. These liquids are solidified into solid tablets orcaplets by freeze drying and solid state extraction techniques. The drugcompounds may be compressed with viscoelastic and thermoelastic sugarsand polymers or effervescent components to produce porous matricesintended for immediate release, without the need of water.

Moreover, the compounds of the present invention can be administered inthe form of nose drops, or metered dose and a nasal or buccal inhaler.The drug is delivered from a nasal solution as a fine mist or from apowder as an aerosol.

The foregoing description of the invention illustrates and describes thepresent invention. Additionally, the disclosure shows and describes onlythe preferred embodiments of the invention but, as mentioned above, itis to be understood that the invention is capable of use in variousother combinations, modifications, and environments and is capable ofchanges or modifications within the scope of the inventive concept asexpressed herein, commensurate with the above teachings and/or the skillor knowledge of the relevant art. The embodiments described hereinaboveare further intended to explain best modes known of practicing theinvention and to enable others skilled in the art to utilize theinvention in such, or other, embodiments and with the variousmodifications required by the particular applications or uses of theinvention. Accordingly, the description is not intended to limit theinvention to the form disclosed herein. Also, it is intended that theappended claims be construed to include alternative embodiments.

1. A compound having the structure as shown below:

wherein, a, b and c may be the same or different and are integers from 0to 12, X equals NH or CHNH₂, R₁ and R₂ can be the same or different andequal to a hydrogen or a straight or branched C₁ to C₂₀ saturated orunsaturated aliphatic; aliphatic amine; an alicyclic; single ormulti-ring aromatic; a single or multi-ring aromatic heterocycle; asingle or multi-ring saturated heterocycle and the halogenated formsthereof.
 2. The compound according to claim 1 wherein said structure hasthe formula:


3. A compound having the structure as shown below:

wherein, a, b and c may be the same or different and are integers from 0to 12; R₁, R₂, R₃, and R₄ can be the same or different and equal to ahydrogen or a straight or branched C₁ to C₂₀ saturated or unsaturatedaliphatic; aliphatic amine; an alicyclic; single or multi-ring aromatic;a single or multi-ring aromatic heterocycle; a single or multi-ringsaturated heterocycle and the halogenated forms thereof.
 4. The compoundaccording to claim 3 wherein said structure has the formula:


5. A pharmaceutical composition useful for treating a disease orcondition which modulates the production, release or activity ofcytokines of inflammatory cells comprising a compound according to claim1 and a pharmaceutically acceptable excipient, diluent or vehicle isdesirable.
 6. A pharmaceutical composition useful for treating a diseaseor condition which modulates the production, release or activity ofcytokines of inflammatory cells comprising a compound according to claim2 and a pharmaceutically acceptable excipient, diluent or vehicle isdesirable.
 7. A pharmaceutical composition useful for treating a diseaseor condition which modulates the production, release or activity ofcytokines of inflammatory cells comprising a compound according to claim3 and a pharmaceutically acceptable excipient, diluent or vehicle isdesirable.
 8. A pharmaceutical composition useful for treating a diseaseor condition which modulates the production, release or activity ofcytokines of inflammatory cells comprising a compound according to claim4 and a pharmaceutically acceptable excipient, diluent or vehicle isdesirable.
 9. A pharmaceutical composition, according to any one ofclaims 5 to 8, useful for treating a disease or condition in which theinhibition of the expression or activity of any or all inflammatorycytokines including but not limited to TNFα, interleukin-1β,interleukin-2, interleukin-6, interleukin-8, interleukin-12,interleukin-18, nitric oxide, granulocyte macrophage-colony stimulatingfactor, granulocyte-colony-stimulating factor,macrophage-colony-stimulating factor, and interferon γ is desirable. 10.The composition of any one of claims 1-4 wherein said excipient, diluentor vehicle is pharmaceutically or cosmetically acceptable.
 11. Thecomposition of any one of claims 1-4 wherein said excipient, diluent orvehicle is for topical or intra-aural administration.
 12. Thecomposition of any one of claims 1-4 formulated for intravenous,subcutaneous, intramuscular, intracranial, intraperitoneal, topical,transdermal, intravaginal, intranasal, intrabronchial, intracranial,intraocular, intraaural, rectal, or parenteral administration.
 13. Amethod of treating one or more conditions associated with unwanted orundesirable presence or activity of inflammatory cytokines comprisingadministration of a compound according to any one of claims 1-4.
 14. Themethod of claim 13 wherein said administration is systemic.
 15. Themethod of claim 13 wherein said administration is oral.
 16. The methodof claim 13 wherein said administration is via time-release vehicle. 17.A method according to claim 13 wherein said condition is selected fromthe group including but not limited to chronic or acute inflammation,inflammatory bowel disease (including Crohn's disease), ulcerativecolitis, irritable bowel syndrome, autoimmune diseases, rheumatoidarthritis, systemic lupus erythematosus, cutaneous forms of lupus,diabetes, multiple sclerosis, psoriasis, spondyloarthropathies (SpA)including spondylitis, synovitis, psoriatic arthritis and subclinicalgut inflammation and infectious diseases including sepsis, septic shock,endotoxic shock, HIV and other viral infections includingcytomegalovirus, herpes simplex virus, and influenza virus and cancerand other proliferative diseases and disorders.